1
Short-Chain Fatty Acids Regulate Cytokines and Th17/Treg Cells in Human 1
Peripheral Blood Mononuclear Cells in vitro 2
3
4
Authors: 5
M. Asarata c, V. Apostolopoulosa b, T. Vasiljevica O. Donkor a* 6
aAdvanced Food Systems Research Unit, College of Health and Biomedicine, Victoria 7
University, Werribee Campus, P.O. Box 14428, Melbourne, Vic 8001, Australia. 8
bVAConsulting Services, P.O. Box 6437, Melbourne, Vic, 3030, Australia. 9
cFaculty of Veterinary Medicine, Tripoli University P.O. Box 13662, Tripoli, Libya. 10
*Corresponding author: Dr O. N. Donkor; Tel: (+61-3)-9919-8059, Fax: (+61-3)-99198284 11
e-mail: Osaana.Donkor@vu.edu.au 12
2 Abstract
14
Short chain fatty acids (SCFAs) have been recognized as mediators of immune responses, 15
including pathways of cytokine production. In this study, we investigated the immune-16
regulatory effects of SCFAs on human peripheral blood mononuclear cells (PBMCs) from 17
buffy coat of healthy donors. PBMCs were exposed to varying concentrations of individual 18
SCFAs or their mixtures, and, the production of interleukin (IL) IL-1β, 2, 6, 10, IL-19
17 IL-21 IL-23 and transforming growth factor beta 1 (TGF-β1) were assessed. T cell 20
differentiation after exposure to SCFAs was also examined. In comparison to LPS-stimulated 21
cells (controls), SCFAs slightly decreased the production of TGF-β1 and significantly 22
reduced IL-6 production (p < 0.05) and butyrate was more effective than acetate or 23
propionate; non-stimulated cells did not respond to SCFAs. In addition, the viability of 24
PBMCs was not significantly affected. SCFAs particularly butyrate caused the induction of 25
CD4+CD25+ regulatory T cells (Treg) rather than Th17 cells. It is clear that. SCFAs may up-26
regulate the production of anti-inflammatory cytokines in PBMCs resulting in the induction 27
of CD4+CD25+ Treg cells. 28
Key words: Short chain fatty acids, Human peripheral blood mononuclear cells, Cytokines, 29
Immune modulation, T helper cells, Th17 and CD4+CD25+ Treg cells. 30
3 1. Introduction
32
Short chain fatty acids (SCFAs) are a sub-group of fatty acids with 2 to 6 carbon atoms (C2 - 33
C6 mono-carboxylic acids), formed principally from fermentation of prebiotics by anaerobic 34
micro-organisms in the colon (Cummings & Macfarlane, 1991). SCFAs are predominant 35
anions in human colon contents (Sellin, 1999). Relative proportions and amounts of SCFAs 36
vary according to the type of fibre in the diet, type and population of microflora present in the 37
colon and the gut transit time, absorption and utilization by colonic epithelium (Macfarlane & 38
Macfarlane, 2003). Hence, the concentrations of SCFAs in the colon, portal blood and 39
peripheral circulation are different. Total SCFAs in the colon has been estimated to range 40
from 70 - 140 mM with concentration ratios of acetate, propionate and butyrate being, 10, 1.5 41
and 2 mM, respectively (Bergman, 1990; Cummings, Pomare, Branch, Naylor, & 42
Macfarlane, 1987; Sellin, 1999; Topping & Clifton, 2001). Whereas in the portal blood, 43
concentrations are typically higher (0.375 mM) compared to that in peripheral circulation 44
(0.079 mM) (Cummings et al., 1987). Likewise, Meijer, de Vos, and Priebe (2010) reported 45
the total concentration of SCFAs in human peripheral blood to ranged from 0.050 – 0.1 mM 46
and in portal blood, about 0.3 – 0.450 mM. 47
SCFAs have been shown to have beneficial effect on human health; they are the favoured 48
fuel of the colonic epithelium and are vital for intestinal and epithelial barrier functions 49
(Adom & Nie, 2013; Scheppach, 1994). Lack of source of energy may lead to diminished 50
integrity of epithelial function resulting in bowel disorders such as bowel inflammation 51
(Harig, Soergel, Komorowski, & Wood, 1989; Wong & Jenkins, 2007). Furthermore, SCFAs 52
stimulate colonic blood flow and enhance fluid and electrolytes uptake such as calcium 53
absorption (Roy, Kien, Bouthillier, & Levy, 2006). Recent interest in SCFAs benefits have 54
focused on their regulatory effects on immune responses by affecting the immune cells 55
4
pathological conditions such as inflammatory bowel disease (IBD), possibly via their effect 57
on immune responses (Tedelind, Westberg, Kjerrulf, & Vidal, 2007; Vernia et al., 1995; 58
Vinolo et al., 2011). However, the mechanism of action is not clearly demonstrated but it has 59
been reported that SCFAs may trigger cellular receptors such as G protein coupled receptors 60
(GPCRs), (GPR41 and GPR43), of immune cells and subsequently initiate the immune 61
response (Bindels, Dewulf, & Delzenne, 2013; Brown et al., 2003; Le Poul et al., 2003; 62
Maslowski et al., 2009; Masui et al., 2013). Moreover, studies have suggested that butyrate 63
and other SCFAs have inhibitory effects on the nuclear factor kappa B (NF-κB) signalling 64
(Liu et al., 2012; Luhrs et al., 2001; Segain et al., 2000; Tedelind et al., 2007) and histone 65
deacetylase (HDAC) (Aoyama, Kotani, & Usami, 2010; Waldecker, Kautenburger, 66
Daumann, Busch, & Schrenk, 2008). SCFAs may regulate cytokine production (Cavaglieri et 67
al., 2003; Masui et al., 2013; Yin, Laevsky, & Giardina, 2001), and, recently, butyrate and 68
propionate have been noted to promote peripheral regulatory T cell (Treg)differentiation 69
which might contribute to immune homeostasis (Arpaia et al., 2013). 70
PBMCs compose mainly of lymphocytes, macrophages and monocytes. The lymphocyte 71
population in healthy human adults consists of approximately 60% T cells (CD4+ and CD8+) 72
and 35% B cells, natural killer cells (NK), macrophage and monocytes (Minoprio, 2000; 73
Plebanski, 2002). PBMCs have been widely used in immunological and pharmaceutical 74
studies probably due to their properties, which relate to sophisticated immune cells. The cells 75
are co-cultured with different types of immune suppressant or stimulant drugs to study and 76
determine their efficiencies using different parameters of immune responses such as the 77
release of immune mediators (cytokines) (Ramachandran et al., 2012). T lymphocytes can be 78
functionally distinguished into cytotoxic (Tc) and helper (Th) cells. Human naive CD4+ T 79
helper cells can be divided into different functional subsets Th1, Th2, Th17, and Treg cells 80
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2012). Th1 cells express T-box transcription factor (Tbet-2) and release IL2, TNFα and IFNγ 82
cytokines and act against intracellular pathogens. Th2 express GATA3, produce anti-83
inflammatory cytokines such as IL-1 receptor antagonist (IL-1ra), IL-4, IL-5, IL-6, IL-10 and 84
IL-13, and stimulate humoral immune responses against extracellular pathogens. Th17 cells 85
express the up-regulation of transcription factors, such as RORγt and STAT-3, release pro-86
inflammatory cytokines IL-17 and play a role in organ-specific autoimmune diseases 87
(Thomas Korn, Oukka, Kuchroo, & Bettelli, 2007). Treg cells express FoxP3 and produce IL-88
10 and TGF-β cytokines, promote tolerance to self and non-pathogenic antigens, suppress 89
amplitude immune and inflammatory responses, drive and modulate immune responses, and 90
abrogate autoimmune diseases (Wan & Flavell, 2009). 91
Mature CD4+ Th cells are also involved in autoimmunity, during T cell receptor (TCR) 92
activation in a particular cytokine milieu, naive CD4 T cells may differentiate into one of 93
several lineages of Th cells, including Th1, Th2, Th17, and induce Treg cells (iTreg) as 94
defined by their pattern of cytokine production and function. Imbalance of either pattern of 95
Th1 or Th2 cells may cause autoimmune disease e.g. a dysregulation of T helper cell 96
phenotype in favour of Th1 appears to underlie Crohn’s disease, a form of IBD (Dionne, 97
Ruemmele, & Seidman, 2004). Along with Th1 and Th2 cells, (FoxP3) Treg and Th17 cells 98
have been recognized as significant players in immune balance (Brand, 2009). The effect of 99
SCFAs on Treg cells may subsequently regulate the induction of Th1, Th2 and Th17 and 100
maintaining the immune homeostasis, thus SCFAs may be valuable to maintain the immune 101
homeostasis and prevent of chronic inflammation (Bailon et al., 2010; Vinolo et al., 2011). 102
SCFAs particularly butyrate have been known to modulate immune responses (Cox et al., 103
2009; Vinolo et al., 2011). However, little is known about the effects of SCFAs on human 104
PBMCs. Herein we evaluated the regulatory effects of SCFAs in PBMCs and the induction of 105
6
inflammatory cytokines with PBMCs in the presence or absence of LPS stimulation was also 107
examined. 108
2. Material and methods 109
2.1.Chemicals and reagent 110
Biological grade acetate, propionate, butyrate,purified LPS from Escherichia coli O111:B4 111
and growth medium Roswell Park Memorial Institute medium (RPMI-1640) were purchased 112
from Sigma (Sigma-Aldrich, Sydney, Australia). Ficoll-Paque™ Plus was from GE 113
Healthcare (GE Healthcare, Bio-Sciences, Uppsala, Sweden). Antibiotic-Antimycotic 114
solution and Fetal bovine serum (FBS) were acquired from Gibco Life Technologies 115
(Gibco®Life Technologies, Mulgrave, Australia). Phosphate-Buffered Saline (1X) pH 7.4, 116
(PBS) was from Invitrogen (Invitrogen Pty Ltd., Mount Waverly, Victoria, Australia),The 117
Buffy coat was provided by the Australian Red Cross Blood Services, Melbourne, Australia, 118
Other reagents and chemicals were of biotechnological and molecular-biology grade from 119
Sigma-Aldrich unless otherwise stated. 120
2.2.Isolation of human PBMCs from buffy coat using Ficoll gradient 121
In order to meet the requirements of the National Health and Medical Research Council 122
“National Statement on Ethical Conduct in Human Research” (National Health and Medical 123
Research Council, 2007), the Human research ethics of the proposed project was accepted 124
and approved by the Chair of the Faculty of Health, Engineering, and Science, Victoria 125
University Human Research Ethics Committee. A contract agreement was also conducted 126
with Australian Red Cross Blood Services Melbourne Australia, in order to the supply of 127
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PBMCs were isolated from human buffy coat by Ficoll-Paque gradient according to the 129
method described by (Donkor et al., 2012) with minor modifications. Briefly, individual 130
buffy coat (60 mL) was diluted with an equal volume of phosphate buffer saline (PBS) and 131
layered on Ficoll-Paque Plus (GE Healthcare, Bio-Sciences, Uppsala, Sweden). Cells at the 132
interphase were collected following centrifugation (680 g, 25 min, 18°C) (Sorvall® RT7 133
centrifuge; DuPont, Newtown, CT, USA). Separated layers PBMC were washed twice in cold 134
PBS, and following centrifugation (680 g, 10 min, 18°C). To lyse any remaining red blood 135
cells, the pellet was resuspended in 5 mL red blood cell lysing buffer; Ammonium-Chloride-136
Potassium (ACK) (Gibco® ACK Life Technologies) and incubated for 8 min at room 137
temperature. The volume was then adjusted to 35 mL using sterile PBS then centrifuged 138
(680 g, 10 min, 18°C). Following two subsequent washes, the cell pellet was resuspended in 139
RPMI1640 medium supplemented with 10% FBS and 1% of Antibiotic-Antimycotic solution 140
for co-culture and stimulation. 141
2.3.Co-culture and stimulation of PBMCs by SCFAs
142
Human PBMCs were seeded in flat bottom 6-well tissue culture plates (Corning, Sigma) at 143
final concentration 1 × 106 cells/mL RPMI1640 medium per well, either in RPMI1640 144
medium alone or with LPS (controls), medium with LPS and acetate, propionate, butyrate or 145
mixed SCFAs. The concentrations of SCFAs were 1, 1.5, 2 mM similar to physiological 146
concentration found in the colon and peripheral circulation (portal vein) (Cummings et al., 147
1987; Topping & Clifton, 2001), similar concentrations were also used in previous studies 148
(Liu et al., 2012; Nancey et al., 2002; Weber & Kerr, 2006). LPS (5 μg/mL) was used for 149
cells stimulation (Chen, Bruns, Donnelly, & Wunderink, 2010; Jansky, Reymanova, & 150
8
To investigate the responses of PBMCs to the stimuli (LPS and/or SCFAs), the cells were 152
stimulated with 5µg/mL LPS alone (Chen et al., 2010), with SCFAs only or a combination of 153
LPS and SCFAs for 48 h. PBMCs were also stimulated with LPS for 24 hour then SCFAs 154
were added and cells further incubated for another 24 hours. 155
2.4.PBMCs viability assay 156
PBMCs viability in presence of LPS and/or SCFAs were assessed using MTS (3-(4,5-157
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner 158
salt) assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, USA) 159
according to manufacture instruction. Briefly, cells were seeded in 96-well plates (Corning, 160
Sigma) at concentration 1 × 103 cells per 100 µL of growth media with or without various 161
stimuli (as above), and then incubated for 48 h at 37°C in a 5% CO2 incubator. The viability 162
was detected by adding 20 µL of MTS solution, followed by 4 h of incubation then 163
absorbance was read at 490 nm using iMark Microplate Absorbance Reader (BIO-RAD, 164
Australia). After subtracting of the background reading, cell viability was calculated as: 165
% viable cell = (Optical density (OD) of SCFAs treated sample / OD of control sample) x 166
100. 167
2.5.ELISA analysis of cytokines 168
Supernatants from stimulated and non-stimulated PBMCs cultures were collected and 169
analysed for cytokines concentrations using BD OptEIA ELISA kits (BD Bioscience, San 170
Diego, CA), including IL-1β, IL-2, IL-6, IL-10, IL-17, IL-21 TGF-β1 and IL-23. The 171
detection procedures were performed in accordance with the manufacturer’s instructions. 172
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cytokine response minus background (pg/ml) of each treatment from triplicate wells, plus or 174
minus the standard error of the mean. 175
2.6.Measurement of nitric oxide production 176
Nitric oxide (NO) production from stimulated PBMCs and non-stimulated PBMCs was 177
measured spectrophotometrically using Griess Reaction Assay (Promiga, Auburn, Australia) 178
according to the manufacture’s instruction. Briefly, after co-culture and stimulation of 179
PBMCs (as above), 50 µL aliquots of supernatant were incubated with 100 µL Griess reagent 180
(50 µL of 1% sulfanilamide in 0.1 M HCl and 50 µL of 0.1% N-1-napthylethylenediamine 181
dihydrochloride) for 10 min at room temperature. Absorbance was read at 550 nm using 182
iMark Microplate Absorbance Reader (BIO-RAD, Australia) and results were calculated 183
based on a NaNO2 standard curve. 184
2.7.Flow Cytometry Analysis of Th17 and Treg cells 185
Activated PBMCs were collected and analysed for induction of Treg/Th17 cells. Briefly, 1 186
mL suspension PBMCs culture was collected and centrifuged at 500g for 10 min, cell pellet 187
was washed twice using fluorescence activated cell sorting (FACS) buffer (PBS + 2% FBS) 188
and the suspension was centrifuged again at 500g for 10 min. PBMCs were re-suspended at 1 189
× 106 cells/ml in FACS buffer and surface marker staining was performed using fluorescein 190
isothiocynate (FITC)-labelled anti-human CD4, allophycocyanin labelled anti-human 191
CD25/CD3 (Becton-Dickinson), peridinin chlorophyll protein (PerCP)-labelled anti-human 192
CD3 (Biolegend, San Diego, CA, USA) and PerCP cyanine (Cy)5.5-labelled anti-human 193
CCR6 (CD196). Intracellular staining was performed using phycoerythrin (PE)-labelled anti-194
human FoxP3/RORγt (BD Pharmingen and R&D Systems, Minneapolis, MN, USA, 195
respectively), according to the manufacturer’s instructions. Samples were read using a BD 196
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Biosciences). Analysis was performed using Gatelogic version 3.07 software (Inivai, 198
Victoria, Australia). Absolute numbers of Treg and Th17 cells were calculated as a 199
percentage of the total lymphocyte number (Donkor et al., 2012). 200
2.8.Statistical analysis 201
The results obtained were analysed as a split plot in time design with three main factors: 202
SCFAs (Three fatty acids) and their doses (3 dose levels) as the main plot and time (two time 203
frame 24 and 48) LPS was used as stimulant. The statistical evaluations of the data were 204
performed using the general linear model (GLM) (SAS/STAT., 1996). Significant differences 205
between treatments were tested by analysis of variance (ANOVA) followed by a comparison 206
between treatments performed by Fisher’s least significant difference (LSD) between each 207
sample with a level of significance of (p < 0.05). Data are expressed as mean and standard 208
deviation of triplicate measures determined in 5 independent experiments. 209
3. Results 210
3.1.PBMCs viability, proliferation and NO production 211
The MTS assay of PBMC viability and proliferation with or without different stimuli is 212
shown in Figure 1. Proliferation of PBMC were potentiated in the presence of LPS in all 213
samples compared with non LPS-stimulated PBMCs. Relatively similar effects resulted in the 214
presence of each SCFA with maximum proliferation observed in the presence of butyrate 215
followed by mixed SCFAs over 48 h.Similar LPS effect on PBMCs proliferation was 216
reported (Jansky et al., 2003), and SCFAs did not induce cell apoptosis at used concentration. 217
Production of NO was enhanced in the presence of LPS, whereas the addition of SCFAs 218
induced remarkable reduction (p < 0.05) of NO (Fig. 2). After 48 h of incubation, NO was 219
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with LPS-stimulated cells, However the inhibition of NO in the SCFA mixture was similar to 221
that of butyrate. 222
3.2.Production of cytokines 223
Figure 3 (A-H) show cytokines (IL-β1, IL-2, IL-6, IL-17, IL-21 IL-23 and TGF-β1) produced 224
by PBMCs in the presence of LPS compared with non-stimulated PBMCs, the combination 225
of LPS and SCFAs or SCFAs only. Results show significant (p < 0.05) decrease in IL-β1, IL-226
2, IL-6, IL-17 and IL-21, with a slight reduction of IL-23 and TGF-β1 in the presence of 227
SCFAs, compared to cells stimulated with LPS only. However, IL-10 secretion was not 228
affected by LPS compared to non LPS-stimulated cells but it was increased in the presence 229
of SCFAs particularly in butyrate (Säemann et al., 2000). We have also noted similar results 230
when PBMCs stimulated with LPS for 24 h followed by addition of SCFAs and incubated for 231
another 24 hours (Table 1). 232
3.3.CD4+ CD25; Th17 and Treg populations 233
Flow cytometric analysis of LPS and SCFAs stimulated PBMCs showed increased CD4+ 234
CD25+ Treg and Th17 cells compared to unstimulated cells (Fig. 4). LPS aloneincreased the 235
Th17 cell population compared to Treg cells after 48 h of incubation. In the presence of 236
SCFAs particularly butyrate, the relative proportion of Th17 and Treg showed slight increase 237
of Treg cells compared to Th17 (Fig. 4). The addition of SCFAs along with LPS at 0 time or 238
after the pre-stimulated PBMCs with LPS, showed comparatively similar up-regulation of 239
Treg cells after 24 h compared to unstimulated cells (control). Butyrate was more effective in 240
induction of Treg cells than acetate or propionate. The addition of mixed SCFAs did not 241
induce augmentation of the effects as compared with each SCFA. However, their effects 242
were concentration-dependent; 2 mM of SCFAs was more effective in up-regulation of Treg 243
12 4. Discussion
245
Despite most of intestinal SCFAs being used by intestinal epithelium cells, a considerable 246
amount of acetate, propionate, and butyrate are absorbed into the blood and exert their effects 247
at peripheral tissue level beyond the digestive system through regulation of immune 248
responses (Matsumoto et al., 2006). Consequently, we assumed that SCFAs might have 249
regulatory effects on peripheral PBMCs. Therefore, in the current study, we used in vitro 250
LPS-stimulated PBMCs of healthy donor to compare the anti-inflammatory effect of acetate, 251
propionate, and butyrate and regulation of Th17 and Treg balance. It was found that SCFAs 252
reduced the production of pro-inflammatory factors including IL-1β, IL-6, IL-17 and NO 253
whereas they enhanced the production of anti-inflammatory mediators such as 10, and IL-254
2. Furthermore, SCFAs affected gene expression of T helper cells possibly through their 255
effect on immune mediators and growth factors such as IL-6 and TGF-β1, which might have 256
led to the up-regulation of Treg cells. Interestingly, SCFAs exhibit these effects mainly in 257
LPS-stimulated PBMCs whereas non LPS-stimulated cells were not affected by SCFAs. 258
4.1.Effect of SCFAs on cells viability 259
To determine whether SCFAs exert their effects through stimulation of PBMCs but not via 260
induce cellular death, MTS assay was conducted after co-culturing of PBMCs with LPS and 261
SCFAs (Fig. 1). Cell growth was slightly potentiated in the presence of LPS and SCFAs 262
particularly butyrate or mixed SCFAs, indicating that viability of cells was not affected by 263
experimental conditions. Furthermore, slight proliferation of PBMCs following LPS exposure 264
in all samples indicated that the proliferation was mainly due to stimulation effect of LPS and 265
that the SCFAs only promoted the LPS activated cell proliferation. The induction of cell 266
proliferation indicate that LPS was able to trigger cell response and initiate the immune 267
13
cells, monocytes, and macrophages. This was expected as LPS is known to induce a 269
macrophage-dependent immune response through activation of NF-κB transcription factor, 270
subsequently enhancing proliferation and release of immune factors from PBMCs (Martich, 271
Boujoukos, & Suffredini, 1993; Sharif, Bolshakov, Raines, Newham, & Perkins, 2007). In 272
our study, acetate, propionate and butyrate did not cause apoptosis in PBMCs but promoted 273
the proliferation of LPS-stimulated PBMCs. Different effects of SCFAs on proliferation of 274
PBMCs and other similar cells have been highlighted (Meijer et al., 2010). A study on the 275
effect of SCFAs in mouse macrophage cell line RAW264.7 reported that viability of cells 276
was not affected when incubated with 0 - 1.2 mM SCFAs (Liu et al., 2012). Similarly, an 277
earlier study demonstrated that unstimulated PBMCs were not affected by SCFAs at a 278
physiological level (Cox et al., 2009). On the other hand some studies showed that butyrate 279
caused apoptosis in antigen stimulated T cells and macrophages (Bailon et al., 2010; Kurita-280
Ochiai, Fukushima, & Ochiai, 1999). For example, 2 mM of butyric acid induced inhibition 281
of proliferation after concanavalin-A stimulated porcine PBMCs (Weber & Kerr, 2006). 282
Another study revealed that lymphocytes proliferation was inhibited due to 1.5 mM of 283
butyrate but not acetate or propionate at the same concentration (Cavaglieri et al., 2003). 284
These different effects of SCFAs might be due to the using of different concentrations, 285
sources of PBMCs and method of stimulation (Meijer et al., 2010). 286
4.2.Effect of SCFAs on cytokine release 287
Lymphocytes besides inflammatory cells are involved in immune and inflammatory 288
responses. They interact with each other via release of cytokines and expression of cytokine 289
receptors in response to stimuli (Gruys, Toussaint, Niewold, & Koopmans, 2005). Cytokines 290
can act as positive or negative regulators of immune responses and maintain lymphocyte 291
balance. Nevertheless, the activity of cytokines are dependent upon each other along with 292
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Schmidt-Arras, & Rose-John, 2011). Factors affecting cytokine network may subsequently 294
influence the inflammatory or immune response. Among these factors, SCFAs which have 295
been recognized as regulators of immune response through different mechanisms, such as 296
directly affecting the immune cells by binding to specific receptors GPCR (Le Poul, et al., 297
2003), inhibition of HDAC (Davie, 2003), suppression of activation of the transcription 298
factor NF-κB (Segain et al., 2000) and revelling different anti-inflammatory activities via 299
regulation of cytokines secretion by immune cells (Cox et al., 2009; Saulnier, Spinler, 300
Gibson, & Versalovic, 2009). 301
The current study highlights the effect of SCFAs on secretion of cytokines that are involved 302
in T cell differentiation into either Th17 or Treg cells besides the main cytokines that are 303
released by Th17 and Treg. Stimulation of PBMCs with LPS alone lead to an increased 304
production of IL-6 and TGF-β as LPS is a known stimulant to induce the secretion of pro-305
inflammatory cytokines (Jansky et al., 2003; Martich et al., 1993; Sharif et al., 2007). The 306
combination of LPS and SCFAs particularly butyrate decreased IL-6 and marginally reduced 307
TGF-β1. These effects were similar for either SCFAs added to 24 h pre-stimulated PBMCs or 308
at 0 time of stimulation (Table 1 and Fig. 3). The reduction of IL-6 and slightly reduced TGF-309
β1concentration in the pre-stimulated PBMCs was probably due to the neutralizing and 310
inhibiting effects of SCFAs on pro-inflammatory condition in stimulated PBMCs. This might 311
have mediated the secretion of anti-inflammatory cytokines that likely act as immune 312
regulator of pro-inflammatory cytokines (Dinarello, 1997; Opal & DePalo, 2000; Sultani, 313
Stringer, Bowen, & Gibson, 2012). The effect of SCFAs on immune cells depends on the 314
activation status and differentiation stages of effector cells (Cox et al., 2009). Furthermore, 315
incubation of PBMCs in the presence of SCFAs without LPS did not induce changes in the 316
release of tested cytokines an indication that the physiological concentrations of SCFAs had 317
15
TGF-β1 has verity of functions, the exposure of PBMCs to TGF-β1 can generate a variety of 319
cellular processes including inhibition of proliferation, differentiation, migration and 320
apoptosis (Sanchez-Capelo, 2005). TGF-β1 regulates many other growth factors and plays a 321
role in naive cell differentiation based on its concentration and other cytokine environment. 322
TGF-β1 in the presence of IL- 6 , IL-1β, IL-21 or IL-23 drive cell differentiation to Th17 323
cells, subsequently releasing more pro-inflammatory factors (Yoshimura, Suzuki, Sakaguchi, 324
Hanada, & Yasukawa, 2012; Liang Zhou, Chong, & Littman, 2009; Liang Zhou et al., 2007). 325
Little is known about effects of SCFAs on release of TGF-β1 in human PBMCs, in our study 326
important pleiotropic cytokine, TGF-β1 was increased significantly in the presence of LPS 327
but only slight decrease with SCFAs. Increase in the concentration of IL-6 along with TGF-328
β1 could trigger differentiation of CD4+ to Th17 cells. The induction of Th17 cells could be 329
related to the down-regulation of IL-6 but not TGF-β1 since the level of TGF-β1 was not 330
significantly affected by SCFAs. Consistent with our study SCFAs have been reported to 331
supress pro-inflammatory mediators such as TNF-α, IL-6 and enhance the release of anti-332
inflammatory cytokine IL-10 (Meijer et al., 2010; Park, Lee, Lee, Kim, & Kim, 2007). 333
Pleiotropic cytokines TGF-β1 plays important role in regulation of immune response by 334
acting with other cytokines such as IL-2 and IL-10 to promote expression and activation of 335
Treg cells, and released more IL-2 and IL-10, consequently up-regulating anti-inflammatory 336
condition and ameliorates inflammation (Taylor, Verhagen, Blaser, Akdis, & Akdis, 2006). 337
4.3.Differentiation of CD4+ cells 338
Based on the cytokine milieu, activated naïve T helper cells may be differentiated into Th1, 339
Th2, Th17 or Treg phenotypes with different effector roles and cytokine profiles (Broere, 340
Apasov, Sitkovsky, & van Eden, 2011). IL-12 milieu skews CD4+ T helper cells to Th1, IL-4 341
skews CD4+ T helper cells to Th2, TGF-β1 skews CD4+ T helper cells to Treg and IL-6 with 342
16
Kimura & Kishimoto, 2010; Ramgolam, Sha, Jin, Zhang, & Markovic-Plese, 2009). 344
Furthermore other cytokines also play a role in cell differentiation and activation such as IL-2 345
which act with TGF-β1 to drive CD4+ T cells to Treg cells (Campbell & Koch, 2011; Zheng, 346
2013; Ziegler & Buckner, 2009) and TGF-β with IL-1β, IL-21, and IL-23 are implicated in 347
promoting human Th17 differentiation (T. Korn, Bettelli, Oukka, & Kuchroo, 2009; 348
Ramgolam et al., 2009; Yang et al., 2008; Zheng, 2013; Ziegler & Buckner, 2009). In our 349
study the, down regulation of Th17 cells may due to decline of IL-21 and IL-23 rather than 350
reduction of TGF-β which was relatively stable in our study condition (Fig. 3). 351
Treg cells and Th17 cells are two subtypes of CD4+ cells. They play opposing roles in 352
autoimmune inflammatory diseases and immune tolerance, although they share a common 353
differentiation pathway. Imbalance of Treg and Th17 has been established in several 354
autoimmune diseases (Ji et al., 2012). Recent studies in human and mouse CD4+ T cells show 355
dichotomy in the generation and differentiation of Treg cells and Th17 cells (Kimura & 356
Kishimoto, 2010; Zhu, Yamane, & Paul, 2010). The TGF-β1 signalling is described as the 357
co-expression of Foxp3 and RORγ-t (L. Zhou et al., 2008). This signalling depends on other 358
immune factors. For example, TGF-β1 alone enhances Foxp3 expression and inhibits RORγ-t 359
activity, whereas combination of TGF-β1 with either IL-1β and IL-23 or IL-21 and IL-6 360
drives human Th17 differentiation (de Jong, Suddason, & Lord, 2010). This indicates the 361
inner correlation between Th17 and Treg cells through the cytokine milieu (Ji et al., 2012). 362
SCFAs are supposed to have a regulatory influence on inflammatory disorder and ameliorate 363
inflammation in some intestinal inflammatory disorders (Cox et al., 2009). This might be 364
mediated through modulation of cytokine milieu in the medium and expression of Treg and 365
Th17. Our finding exhibited that Foxp3 and RORγ-t expression in T lymphocytes subset 366
population resulted after incubation of LPS stimulated PBMCs with different SCFAs. The 367
17
leading to increased frequency of Th17 and the subsequent release of IL-17 and IL-21. 369
However in the presence of SCFAs, Th17 cell differentiation was likely supressed and 370
favoured cytokine environment for FoxP3 regulatory cell induction leading to enhanced 371
FoxP3 expression. Butyrate was more effective in this regard than acetate and propionate. 372
These findings are consistent with studies suggesting that butyrate showed strong anti-373
inflammatory properties (Cavaglieri et al., 2003; Liu et al., 2012; Meijer et al., 2010; 374
Tedelind et al., 2007) Furthermore, Arpaia et al. (2013) and Furusawa et al. (2013) reported 375
that butyrate promotes the regulation of intestinal Treg generation. However, the effect of 376
SCFAs on T cell phenotypes need more studies. 377
5. Conclusion 378
SCFAs may have in vitro anti-inflammatory and immune regulatory effects through induction 379
of Treg cells and production of anti-inflammatory cytokines. Butyrate showed more 380
regulatory effect than propionate and acetate respectively. Our finding indicates that, SCFAs 381
may have regulatory properties on inflammatory processes via the balance of Th17/Treg cells 382
and pro and anti-inflammatory cytokines. 383
Acknowledgements 384
We gratefully acknowledge the Ministry of High Education of the Libyan Government and 385
Victoria University for their financial and technical support. We thank the Australian Red 386
Cross Services for their supply of blood products and technical supports by Burnet Institute, 387
and we gratefully acknowledge researchers and technical staff at Werribee Campus, Victoria 388
18 Reference
390
Adom, D., & Nie, D. (2013). Regulation of Autophagy by Short Chain Fatty Acids in Colon 391
Cancer Cells. 392
Afzali, B., Lombardi, G., Lechler, R. I., & Lord, G. M. (2007). The role of T helper 17 393
(Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune 394
disease. Clin Exp Immunol, 148(1), 32-46. doi: 10.1111/j.1365-2249.2007.03356.x 395
Aoyama, M., Kotani, J., & Usami, M. (2010). Butyrate and propionate induced activated or 396
non-activated neutrophil apoptosis via HDAC inhibitor activity but without activating 397
GPR-41/GPR-43 pathways. Nutrition, 26(6), 653-661. doi: 398
http://dx.doi.org/10.1016/j.nut.2009.07.006 399
Arpaia, N., Campbell, C., Fan, X., Dikiy, S., van der Veeken, J., Liu, H., Rudensky, A. Y. 400
(2013). Metabolites produced by commensal bacteria promote peripheral regulatory 401
T-cell generation. Nature, 504(7480), 451-455. 402
Bailon, E., Cueto-Sola, M., Utrilla, P., Rodriguez-Cabezas, M. E., Garrido-Mesa, N., 403
Zarzuelo, A., Comalada, M. (2010). Butyrate in vitro immune-modulatory effects 404
might be mediated through a proliferation-related induction of apoptosis. 405
Immunobiology, 215(11), 863-873. doi: 10.1016/j.imbio.2010.01.001 406
Bergman, E. N. (1990). Energy contributions of volatile fatty acids from the gastrointestinal 407
tract in various species. Physiological Reviews, 70(2), 567-590. 408
Bindels, L. B., Dewulf, E. M., & Delzenne, N. M. (2013). GPR43/FFA2: physiopathological 409
relevance and therapeutic prospects. Trends in pharmacological sciences, 34(4), 226-410
232. 411
Brand, S. (2009). Crohn's disease: Th1, Th17 or both? The change of a paradigm: new 412
immunological and genetic insights implicate Th17 cells in the pathogenesis of 413
19
Broere, F., Apasov, S. G., Sitkovsky, M. V., & van Eden, W. (2011). A2 T cell subsets and T 415
cell-mediated immunity Principles of Immunopharmacology (pp. 15-27): Springer. 416
Brown, A. J., Goldsworthy, S. M., Barnes, A. A., Eilert, M. M., Tcheang, L., Daniels, D., 417
Dowell, S. J. (2003). The Orphan G Protein-coupled Receptors GPR41 and GPR43 418
Are Activated by Propionate and Other Short Chain Carboxylic Acids. Journal of 419
Biological Chemistry, 278(13), 11312-11319. doi: 10.1074/jbc.M211609200 420
Campbell, D. J., & Koch, M. A. (2011). Phenotypical and functional specialization of 421
FOXP3+ regulatory T cells. Nat Rev Immunol, 11(2), 119-130. doi: 10.1038/nri2916 422
Cavaglieri, C. R., Nishiyama, A., Fernandes, L. C., Curi, R., Miles, E. A., & Calder, P. C. 423
(2003). Differential effects of short-chain fatty acids on proliferation and production 424
of pro- and anti-inflammatory cytokines by cultured lymphocytes. Life Sciences, 425
73(13), 1683-1690. doi: http://dx.doi.org/10.1016/S0024-3205(03)00490-9 426
Chen, J., Bruns, A. H., Donnelly, H. K., & Wunderink, R. G. (2010). Comparative in vitro 427
stimulation with lipopolysaccharide to study TNFalpha gene expression in fresh 428
whole blood, fresh and frozen peripheral blood mononuclear cells. J Immunol 429
Methods, 357(1-2), 33-37. 430
Cox, M. A., Jackson, J., Stanton, M., Rojas-Triana, A., Bober, L., Laverty, M., Jenh, C. H. 431
(2009). Short-chain fatty acids act as antiinflammatory mediators by regulating 432
prostaglandin E(2) and cytokines. World J Gastroenterol, 15(44), 5549-5557. 433
Cummings, J. H., & Macfarlane, G. T. (1991). The control and consequences of bacterial 434
fermentation in the human colon. Journal of Applied Microbiology, 70(6), 443-459. 435
doi: 10.1111/j.1365-2672.1991.tb02739.x 436
Cummings, J. H., Pomare, E. W., Branch, W. J., Naylor, C. P., & Macfarlane, G. T. (1987). 437
Short chain fatty acids in human large intestine, portal, hepatic and venous blood. 438
20
Davie, J. R. (2003). Inhibition of Histone Deacetylase Activity by Butyrate. The Journal of 440
Nutrition, 133(7), 2485S-2493S. 441
de Jong, E., Suddason, T., & Lord, G. M. (2010). Translational mini-review series on Th17 442
cells: development of mouse and human T helper 17 cells. Clin Exp Immunol, 159(2), 443
148-158. doi: 10.1111/j.1365-2249.2009.04041.x 444
Dinarello, C. A. (1997). Proinflammatory and anti-inflammatory cytokines as mediators in 445
the pathogenesis of septic shock. CHEST Journal, 112(6_Supplement), 321S-329S. 446
Dionne, S., Ruemmele, F. M., & Seidman, E. G. (2004). Immunopathogenesis of 447
inflammatory bowel disease: role of cytokines and immune cell-enterocyte 448
interactions. 449
Donkor, O. N., Ravikumar, M., Proudfoot, O., Day, S. L., Apostolopoulos, V., Paukovics, G., 450
Gill, H. (2012). Cytokine profile and induction of T helper type 17 and regulatory T 451
cells by human peripheral mononuclear cells after microbial exposure. Clinical & 452
Experimental Immunology, 167(2), 282-295. doi: 10.1111/j.1365-2249.2011.04496.x 453
Gruys, E., Toussaint, M. J., Niewold, T. A., & Koopmans, S. J. (2005). Acute phase reaction 454
and acute phase proteins. J Zhejiang Univ Sci B, 6(11), 1045-1056. doi: 455
10.1631/jzus.2005.B1045 456
Harig, J. M., Soergel, K. H., Komorowski, R. A., & Wood, C. M. (1989). Treatment of 457
diversion colitis with short-chain-fatty acid irrigation. New England Journal of 458
Medicine, 320(1), 23-28. 459
Jansky, L., Reymanova, P., & Kopecky, J. (2003). Dynamics of cytokine production in 460
human peripheral blood mononuclear cells stimulated by LPS or infected by Borrelia. 461
21
Ji, L., Zhan, Y., Hua, F., Li, F., Zou, S., Wang, W., Cheng, Y. (2012). The Ratio of 463
Treg/Th17 Cells Correlates with the Disease Activity of Primary Immune 464
Thrombocytopenia. PLoS ONE, 7(12), e50909. doi: 10.1371/journal.pone.0050909 465
Kimura, A., & Kishimoto, T. (2010). IL-6: Regulator of Treg/Th17 balance. European 466
Journal of Immunology, 40(7), 1830-1835. doi: 10.1002/eji.201040391 467
Korn, T., Bettelli, E., Oukka, M., & Kuchroo, V. K. (2009). IL-17 and Th17 Cells Annual 468
Review of Immunology (Vol. 27, pp. 485-517). Palo Alto: Annual Reviews. 469
Korn, T., Oukka, M., Kuchroo, V., & Bettelli, E. (2007). Th17 cells: Effector T cells with 470
inflammatory properties. Seminars in Immunology, 19(6), 362-371. doi: 471
http://dx.doi.org/10.1016/j.smim.2007.10.007 472
Kurita-Ochiai, T., Fukushima, K., & Ochiai, K. (1999). Lipopolysaccharide Stimulates 473
Butyric Acid-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells. 474
Infection and Immunity, 67(1), 22-29. 475
Le Poul, E., Loison, C., Struyf, S., Springael, J.-Y., Lannoy, V., Decobecq, M.-E., Detheux, 476
M. (2003). Functional Characterization of Human Receptors for Short Chain Fatty 477
Acids and Their Role in Polymorphonuclear Cell Activation. Journal of Biological 478
Chemistry, 278(28), 25481-25489. doi: 10.1074/jbc.M301403200 479
Liu, T., Li, J., Liu, Y., Xiao, N., Suo, H., Xie, K., Wu, C. (2012). Short-chain fatty acids 480
suppress lipopolysaccharide-induced production of nitric oxide and proinflammatory 481
cytokines through inhibition of NF-κB pathway in RAW264. 7 cells. Inflammation, 482
35(5), 1676-1684. 483
Luckheeram, R. V., Zhou, R., Verma, A. D., & Xia, B. (2012). CD4+T Cells: Differentiation 484
and Functions. Clinical and Developmental Immunology, 2012, 12. doi: 485
22
Luhrs, H., Gerke, T., Boxberger, F., Backhaus, K., Melcher, R., Scheppach, W., & Menzel, 487
T. (2001). Butyrate inhibits interleukin-1-mediated nuclear factor-kappa B activation 488
in human epithelial cells. [Research Support, Non-U S Gov't]. Dig Dis Sci, 46(9), 489
1968-1973. 490
Macfarlane, S., & Macfarlane, G. T. (2003). Regulation of short-chain fatty acid production. 491
Proceedings of the Nutrition Society, 62(01), 67-72. doi: doi:10.1079/PNS2002207 492
Martich, G. D., Boujoukos, A. J., & Suffredini, A. F. (1993). Response of man to endotoxin. 493
[Review]. Immunobiology, 187(3-5), 403-416. 494
Maslowski, K. M., Vieira, A. T., Ng, A., Kranich, J., Sierro, F., Di, Y., Mackay, C. R. (2009). 495
Regulation of inflammatory responses by gut microbiota and chemoattractant receptor 496
GPR43. [10.1038/nature08530]. Nature, 461(7268), 1282-1286. doi: 497
http://www.nature.com/nature/journal/v461/n7268/suppinfo/nature08530_S1.html 498
Masui, R., Sasaki, M., Funaki, Y., Ogasawara, N., Mizuno, M., Iida, A., Kasugai, K. (2013). 499
G protein-coupled receptor 43 moderates gut inflammation through cytokine 500
regulation from mononuclear cells. Inflamm Bowel Dis, 19(13), 2848-2856. doi: 501
10.1097/01.MIB.0000435444.14860.ea 502
Matsumoto, N., Riley, S., Fraser, D., Al-Assaf, S., Ishimura, E., Wolever, T., Phillips, A. O. 503
(2006). Butyrate modulates TGF-β1 generation and function: Potential renal benefit 504
for Acacia (sen) SUPERGUM™(gum arabic)? Kidney international, 69(2), 257-265. 505
Meijer, K., de Vos, P., & Priebe, M. G. (2010). Butyrate and other short-chain fatty acids as 506
modulators of immunity: what relevance for health? Curr Opin Clin Nutr Metab 507
Care, 13(6), 715-721. doi: 10.1097/MCO.0b013e32833eebe5 508
Minoprio, P. (2000). Lymphocytes: A Practical Approach (2nd edn) by S.L. Rowland-Jones 509
and A.J. McMichel. Parasitology Today, 16(8), 360. doi:
510
23
Nancey, S., Bienvenu, J., Coffin, B., Andre, F., Descos, L., & Flourie, B. (2002). Butyrate 512
strongly inhibits in vitro stimulated release of cytokines in blood. Dig Dis Sci, 47(4), 513
921-928. 514
Opal, S. M., & DePalo, V. A. (2000). Anti-inflammatory cytokines. CHEST Journal, 117(4), 515
1162-1172. 516
Park, J. S., Lee, E. J., Lee, J. C., Kim, W. K., & Kim, H. S. (2007). Anti-inflammatory effects 517
of short chain fatty acids in IFN-gamma-stimulated RAW 264.7 murine macrophage 518
cells: involvement of NF-kappaB and ERK signaling pathways. Int 519
Immunopharmacol, 7(1), 70-77. doi: 10.1016/j.intimp.2006.08.015 520
Plebanski, M. (2002). Preparation of Lymphocytes and Identification of Lymphocytes 521
Subpopulations. In S. L. R.-J. a. A. McMichael (Ed.), Lymphocytes A Practical 522
Approach. New York, USA: Oxford University Press. 523
Ramachandran, H., Laux, J., Moldovan, I., Caspell, R., Lehmann, P. V., & Subbramanian, R. 524
A. (2012). Optimal thawing of cryopreserved peripheral blood mononuclear cells for 525
use in high-throughput human immune monitoring studies. Cells, 1(3), 313-324. 526
Ramgolam, V. S., Sha, Y., Jin, J., Zhang, X., & Markovic-Plese, S. (2009). IFN-β Inhibits 527
Human Th17 Cell Differentiation. The Journal of Immunology, 183(8), 5418-5427. 528
doi: 10.4049/jimmunol.0803227 529
Roy, C. C., Kien, C. L., Bouthillier, L., & Levy, E. (2006). Short-chain fatty acids: ready for 530
prime time? Nutr Clin Pract, 21(4), 351-366. 531
Säemann, M. D., Böhmig, G. A., Österreicher, C. H., Burtscher, H., Parolini, O., Diakos, C., 532
Zlabinger, G. J. (2000). Anti-inflammatory effects of sodium butyrate on human 533
monocytes: potent inhibition of IL-12 and up-regulation of IL-10 production. The 534
24
Sanchez-Capelo, A. (2005). Dual role for TGF-beta1 in apoptosis. Cytokine Growth Factor 536
Rev, 16(1), 15-34. doi: 10.1016/j.cytogfr.2004.11.002 537
SAS/STAT. (1996). SAS/STAT® Software: Changes and Enhancements: Through Release 538
6.11: SAS Institute Cary, North Carolina. 539
Saulnier, D. M. A., Spinler, J. K., Gibson, G. R., & Versalovic, J. (2009). Mechanisms of 540
probiosis and prebiosis: considerations for enhanced functional foods. Current 541
Opinion in Biotechnology, 20(2), 135-141. doi: 10.1016/j.copbio.2009.01.002 542
Scheller, J., Chalaris, A., Schmidt-Arras, D., & Rose-John, S. (2011). The pro- and anti-543
inflammatory properties of the cytokine interleukin-6. Biochimica et Biophysica Acta 544
(BBA) - Molecular Cell Research, 1813(5), 878-888. doi: 545
http://dx.doi.org/10.1016/j.bbamcr.2011.01.034 546
Scheppach, W. (1994). Effects of short chain fatty acids on gut morphology and function. 547
Gut, 35(1 Suppl), S35-38. 548
Segain, J. P., Raingeard de la Bletiere, D., Bourreille, A., Leray, V., Gervois, N., Rosales, C., 549
Galmiche, J. P. (2000). Butyrate inhibits inflammatory responses through NFkappaB 550
inhibition: implications for Crohn's disease. Gut, 47(3), 397-403. 551
Sellin, J. H. (1999). SCFAs: The Enigma of Weak Electrolyte Transport in the Colon. News 552
Physiol Sci, 14, 58-64. 553
Sharif, O., Bolshakov, V. N., Raines, S., Newham, P., & Perkins, N. D. (2007). 554
Transcriptional profiling of the LPS induced NF-kappaB response in macrophages. 555
BMC Immunol, 8, 1. 556
Sultani, M., Stringer, A. M., Bowen, J. M., & Gibson, R. J. (2012). Anti-Inflammatory 557
Cytokines: Important Immunoregulatory Factors Contributing to Chemotherapy-558
Induced Gastrointestinal Mucositis. Chemotherapy Research and Practice, 2012, 11. 559
25
Taylor, A., Verhagen, J., Blaser, K., Akdis, M., & Akdis, C. A. (2006). Mechanisms of 561
immune suppression by interleukin-10 and transforming growth factor-beta: the role 562
of T regulatory cells. Immunology, 117(4), 433-442. doi: 10.1111/j.1365-563
2567.2006.02321.x 564
Tedelind, S., Westberg, F., Kjerrulf, M., & Vidal, A. (2007). Anti-inflammatory properties of 565
the short-chain fatty acids acetate and propionate: a study with relevance to 566
inflammatory bowel disease. World J Gastroenterol, 13(20), 2826-2832. 567
Topping, D. L., & Clifton, P. M. (2001). Short-Chain Fatty Acids and Human Colonic 568
Function: Roles of Resistant Starch and Nonstarch Polysaccharides. Physiological 569
Reviews, 81(3), 1031-1064. 570
Vernia, P., Marcheggiano, A., Caprilli, R., Frieri, G., Corrao, G., Valpiani, D., Torsoli, A. 571
(1995). Short-chain fatty acid topical treatment in distal ulcerative colitis. Aliment 572
Pharmacol Ther, 9(3), 309-313. 573
Vinolo, M. A., Rodrigues, H. G., Nachbar, R. T., & Curi, R. (2011). Regulation of 574
inflammation by short chain fatty acids. Nutrients, 3(10), 858-876. doi: 575
10.3390/nu3100858 576
Waldecker, M., Kautenburger, T., Daumann, H., Busch, C., & Schrenk, D. (2008). Inhibition 577
of histone-deacetylase activity by short-chain fatty acids and some polyphenol 578
metabolites formed in the colon. The Journal of Nutritional Biochemistry, 19(9), 587-579
593. doi: http://dx.doi.org/10.1016/j.jnutbio.2007.08.002 580
Wan, Y. Y., & Flavell, R. A. (2009). How diverse--CD4 effector T cells and their functions. J 581
Mol Cell Biol, 1(1), 20-36. 582
Weber, T. E., & Kerr, B. J. (2006). Butyrate differentially regulates cytokines and 583
proliferation in porcine peripheral blood mononuclear cells. Veterinary immunology 584
26
Wong, J. M. W., & Jenkins, D. J. A. (2007). Carbohydrate Digestibility and Metabolic 586
Effects. The Journal of Nutrition, 137(11), 2539S-2546S. 587
Yang, L., Anderson, D. E., Baecher-Allan, C., Hastings, W. D., Bettelli, E., Oukka, M., 588
Hafler, D. A. (2008). IL-21 and TGF-beta are required for differentiation of human 589
T(H)17 cells. Nature, 454(7202), 350-352. doi: 10.1038/nature07021 590
Yin, L., Laevsky, G., & Giardina, C. (2001). Butyrate Suppression of Colonocyte NF-κB 591
Activation and Cellular Proteasome Activity. Journal of Biological Chemistry, 592
276(48), 44641-44646. doi: 10.1074/jbc.M105170200 593
Yoshimura, A., Suzuki, M., Sakaguchi, R., Hanada, T., & Yasukawa, H. (2012). SOCS, 594
inflammation and autoimmunity. [Review]. Frontiers in Immunology, 3. doi: 595
10.3389/fimmu.2012.00020 596
Zheng, S. G. (2013). Regulatory T cells vs Th17: differentiation of Th17 versus Treg, are the 597
mutually exclusive? Am J Clin Exp Immunol, 2(1), 94-106. 598
Zhou, L., Chong, M. M., & Littman, D. R. (2009). Plasticity of CD4+ T Cell Lineage 599
Differentiation. Immunity, 30(5), 646-655. 600
Zhou, L., Ivanov, I. I., Spolski, R., Min, R., Shenderov, K., Egawa, T., Littman, D. R. (2007). 601
IL-6 programs TH-17 cell differentiation by promoting sequential engagement of the 602
IL-21 and IL-23 pathways. Nature immunology, 8(9), 967-974. 603
Zhou, L., Lopes, J. E., Chong, M. M., Ivanov, II, Min, R., Victora, G. D., Littman, D. R. 604
(2008). TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing 605
RORgammat function. Nature, 453(7192), 236-240. doi: 10.1038/nature06878 606
Zhu, J., Yamane, H., & Paul, W. E. (2010). Differentiation of effector CD4 T cell populations 607
(*). Annu Rev Immunol, 28, 445-489. doi: 10.1146/annurev-immunol-030409-101212 608
Ziegler, S. F., & Buckner, J. H. (2009). FOXP3 and the regulation of Treg/Th17 609
differentiation. Microbes Infect, 11(5), 594-598. doi: 10.1016/j.micinf.2009.04.002 610
27 List of figures:
612
Fig. 1. 613
MTS assay examining the effects of SCFAs on the viability and proliferation of PBMCs. 614
Cells were sub-cultured in 96 well plates at initial density of 103 cells per 100 μL with or 615
without SCFAs and/or LPS and incubated for 48 h. PBMCs in growth media was set as 616
control. Cells viability (%) = (OD of SCFAs treated sample / OD of control sample) ×100. 617
Ace = acetate, Pro = propionate, But = Butyrate, PBMCs = Peripheral blood monocular cells. 618
Results are expressed as mean of five independent experiments with error bars showing the 619
standard deviation, (p < 0.05). 620
621
Fig. 2. 622
Nitric oxide (NO) concentration (µM) in supernatants of stimulated PBMCs (1× 106 cells/ 623
mL) cultures. PBMCs were sub-cultured for 48 h either in the presence of LPS (5 μg/mL), 624
LPS with each SCFA (acteate, propionate or butyrate) or LPS with the mixed SCFAs. RPMI 625
media only was used as a control. Supernatants were collected after 24 h and 48 h and the 626
level of NO was determined by the Griess reaction. NO levels were significantly different (p 627
< 0.05) from the control values (bars with different letter). NO = Nitric oxide; LPS = 628
Lipopolysaccharide; Ace = acetate; Pro = propionate; But = butyrate; SCFAs = short chain 629
fatty acids. Data represent the mean of five independent experiments with error bars showing 630
the standard deviation. 631
632
Fig. 3. 633
Cytokine concentrations of IL-1β (A), IL-2 (B), IL-6 (C), IL-10 (D), IL-17 (E), TGF-β1 (F), 634
IL-21 (G) and IL-23 (H) in supernatant after PBMCs (1 × 106 cells/mL) were sub-cultured 635
with either acetate, propionate, butyrate or mixed SCFAs for 24 h and 5 μg/mL. LPS was 636
added to each well and incubated for a further 24 h or 48 h. PBMCs with LPS only was set as 637
control. Data represent the mean of five independent experiments with error bars showing the 638
standard deviation, (bars with different letter are significantly different (p < 0.05)). SCFAs = 639
short chain fatty acids; IL = interleukin; TGF = transforming growth factor; Ctrl = control, 640
LPS = Lipopolysaccharide, Ace = acetate, Pro = propionate, But = butyrate, Mix = Mixed 641 SCFAs. 642 643 Fig. 4. 644
Expression of activation marker CD25 on T lymphocytes, plots were gated on CD3 (A), 645
percentage of induced CD25+ forkhead box protein 3 (FoxP3+) (B), and induction of ROR-γt 646
expressing T helper type 17 (Th17) (C) by PBMCs in response to LPS and/or SCFAs. 647
28 Table 1
650
Cytokine levels (pg/mL) produced by PBMCs co-cultured with LPS only for 24 h then SCFAs were added and the cells were incubated for 651
another 24 h. 652
Cytokines T(h) Ctrl LPS Ace Pro But Mix
IL-β1 24 55.04 ±6.3 1587.1 ±203.8 1571.7 ±130.2 1275.2 ±51.2 1601.1± 105.4 1620.1 ±33.0 48 53.13 ±5.8 1828.2 ±166.4 1031.7 ±35.5 1121.2 ±48.4 992.5 ±166.3 1086.3 ±143.0
IL-2 24 17.33 ±0.5 285.13 ±5.1 291.32 ±12.2 287.23 ±8.5 290.1 ±41.7 289.21 ±6.1
48 15.46 ±1.3 346.21 ±21.1 225 ±31.5 234 ±19.8 201.3 ±32.2 222 ±7.6
IL-6 24 59.36 ±3.3 1082.07 ±20.6 1001.21 ±25.7 1013 ±5.1 1014.12 ±83.3 997.39 ±64.6
48 71.41 ±5.2 1097.13 ±11.9 831.3 ±32.4 942 ±25.1 612 ±32.9 741 ±46.2
IL-10 24 71.12 ±4.2 76.18 ±3.6 78.3 ±13.3 88.1 ±1.2 87.23 ±4.8 76.91 ±3.5
48 77.34 ±3.2 74.75 ±2.8 181.2 ±6.4 173.15 ±8.1 26 ±12 263 ±20.8
IL-17 24 74.13 ±5.3 403.17 ±34.1 423.1 ±8.7 411.1 ±21.1 435.2 ±23.0 429.19 ±46.4
48 78.41 ±1.9 432 ±14.1 107.4 ±2.8 172 ±5.3 121 ±10.5 133 ±16.8
IL-21 24 231.12 ±14.4 371.41 ±6.9 372.42 ±95.9 358 ±18.5 364.3 ±28.0 361.1 ±17.1
48 236.71 ±12 391.35 ±19.9 62.21 ±3.3 117.7 ±5.9 61.5 ±9.4 75.13 ±4.0
IL-23 24 81.28 ±3.8 1271 ±89.8 1237 ±175.1 1211.5 ±81.1 1301 ±178.4 1291 ±38.3
48 86.30 ±3.2 1295 ±116.0 920 ±34.1 989 ±32.1 931.2 ±11.7 1011 ±26.8
TGF-β1 24 161.1 ±9.1 1751 ±127.5 1723 ±108.2 1715 ±81.1 1695.4 ±17.7 1699 ±116.5
48 156.3±4.1 1950±38.4 1710±46.1 1608±135.6 1651±31.5 1558±174.0
IL = interleukin; TGF-β1 = transforming growth factor beta 1; Ctrl = control (PBMCs without stimulation), LPS = Lipopolysaccharide, Ace = 653
29 Fig. 1.
655
656 657
92 93 94 95 96 97 98 99 100 101
PBMCs Ace Pro But SCFAs
C
ells
V
ia
b
ilit
y
%
SCFAs (2mM)
LPS
30 Fig. 2.
658
659
0 10 20 30 40 50 60 70 80 90
Control LPS Act Pro But SCFAs
µ
M
o
f N
O
SCFA 2 mM
0 24 48
a a a a a a a a
b b
c c
c c c c
a a a a a
b b
31 Fig. 3.
660
661
662
0 400 800 1200 1600 2000
1 1.5 2 1 1.5 2 1 1.5 2 1 1.5 2
Ctrl LPS Acetate Propionate Butyrate Mixed
IL
-1
β
co
n
cen
tra
ti
o
n
n
g
/m
L
24 48
A
a b
c c
b bc
c c c c c c c
c
0 100 200 300 400
1 1.5 2 1 1.5 2 1 1.5 2 1 1.5 2
Ctrl LPS Acetate Propionate Butyrate Mixed
IL
-2 c
on
ce
n
tr
at
ion
p
g/
m
L
24 48
B
a b
c c c c c c
32 663
664
665
0 200 400 600 800 1000 1200 1400
1 1.5 2 1 1.5 2 1 1.5 2 1 1.5 2
Ctrl LPS Acetate Propionate Butyrate Mixed
IL
-6 c
on
ce
n
tr
at
ion
p
g/
m
L
24 48
C
a b
cd
c c c c c c
d d c
cd cd
0 50 100 150 200 250 300
1 1.5 2 1 1.5 2 1 1.5 2 1 1.5 2
Ctrl LPS Acetate Propionate Butyrate Mixed
IL
-10 c
on
ce
n
tr
at
ion
p
g/
m
L
24 48
D
a a
b b b b b b
d d d d
33 666
667
668
0 100 200 300 400 500
1 1.5 2 1 1.5 2 1 1.5 2 1 1.5 2
Ctrl LPS Acetate Propionate Butyrate Mixed
IL
-17 c
on
ce
n
tr
at
ion
p
g/
m
L
24 48
E
a b
a a c
c c
c c c c
ac ac
ac
0 400 800 1200 1600 2000
1 1.5 2 1 1.5 2 1 1.5 2 1 1.5 2
Ctrl LPS Acetate Propionate Butyrate Mixed
T
GF
-β
1
con
ce
n
tr
at
ion
p
g/
m
L
24 48
F
a b
b b b
34 669
670
671
0 100 200 300 400 500
1 1.5 2 1 1.5 2 1 1.5 2 1 1.5 2
Ctrl LPS Acetate Propionate Butyrate Mixed
IL
-21 c
on
ce
n
tr
at
ion
p
g/
m
L
24 48
G
a b
c c c c c c c c c d d d
0 300 600 900 1200 1500
1 1.5 2 1 1.5 2 1 1.5 2 1 1.5 2
Ctrl LPS Acetate Propionate Butyrate Mixed
IL
-23 c
on
ce
n
tr
at
ion
p
g/
m
L
24 48
H
a b
35 Fig. 4.
